Small aperture large electrode cell

ABSTRACT

A nanopore cell includes a conductive layer and a working electrode disposed above the conductive layer and at the bottom of a well into which an electrolyte may be contained, such that at least a portion of a top base surface area of the working electrode is exposed to the electrolyte. The nanopore cell further includes a first insulating wall disposed above the working electrode and surrounding a lower section of a well, and a second insulating wall disposed above the first insulating wall and surrounding an upper section of the well, forming an overhang above the lower section of the well. The upper section of the well includes an opening that a membrane may span across, and wherein a base surface area of the opening is smaller than the at least a portion of the top base surface area of the working electrode that is exposed to the electrolyte.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/948,401, filed Sep. 16, 2020, which is a divisional application ofU.S. patent application Ser. No. 14/841,127, filed Aug. 31, 2015 andtitled “SMALL APERTURE LARGE ELECTRODE CELL,” each of which is hereinincorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

Advances in micro-miniaturization within the semiconductor industry inrecent years have enabled biotechnologists to begin packingtraditionally bulky sensing tools into smaller and smaller form factors,onto so-called biochips. It would be desirable to develop techniques forbiochips that make them more robust, efficient, and cost-effective.

BRIEF DESCRIPTION OF THE DRAWINGS

Various embodiments of the invention are disclosed in the followingdetailed description and the accompanying drawings.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip.

FIG. 2 illustrates an embodiment of a cell 200 performing nucleotidesequencing with the Nano-SBS technique.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags.

FIG. 5 illustrates an embodiment of a circuitry 500 in a cell of ananopore based sequencing chip.

FIG. 6 illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state.

FIG. 7A illustrates an additional embodiment of a circuitry 700 in acell of a nanopore based sequencing chip, wherein the voltage appliedacross the nanopore can be configured to vary over a time period duringwhich the nanopore is in a particular detectable state.

FIG. 7B illustrates an additional embodiment of a circuitry 701 in acell of a nanopore based sequencing chip, wherein the voltage appliedacross the nanopore can be configured to vary over a time period duringwhich the nanopore is in a particular detectable state.

FIG. 7C illustrates a double layer that is formed at any interfacebetween a conductive electrode and an adjacent liquid electrolyte. Inthe example shown, the electrode surface is negatively charged,resulting in the accumulation of positively charged species in theelectrolyte. In another example, the polarity of all charges shown maybe opposite to the example shown.

FIG. 7D illustrates a pseudocapacitance effect that can be formed,simultaneously with the formation of a double-layer as in FIG. 7C, at aninterface between a conductive electrode and an adjacent liquidelectrolyte.

FIG. 8 illustrates an embodiment of a process 800 for analyzing amolecule inside a nanopore, wherein the nanopore is inserted in amembrane.

FIG. 9 illustrates an embodiment of a plot of the voltage applied acrossthe nanopore versus time when process 800 is performed and repeatedthree times.

FIG. 10 illustrates an embodiment of the plots of the voltage appliedacross the nanopore versus time when the nanopore is in differentstates.

FIG. 11 illustrates an embodiment of a non-faradaic electrochemical cell1100 of a nanopore based sequencing chip that includes a TiN workingelectrode with increased electrochemical capacitance.

FIG. 12 illustrates a top view of a plurality of circular openings 1202of a plurality of wells in a nanopore based sequencing chip.

FIG. 13 illustrates an embodiment of a process for constructing anon-faradaic electrochemical cell of a nanopore based sequencing chipthat includes a TiN working electrode with increased electrochemicalcapacitance.

FIGS. 14A-E illustrate an embodiment of a process 1400 for constructinga non-faradaic electrochemical cell that has a smaller aperture openingto a well for the formation of a lipid bilayer with a smaller basesurface area and a working electrode with a larger base surface area.

FIGS. 15A-F illustrate an embodiment of a process 1500 for constructinga non-faradaic electrochemical cell that has a smaller aperture openingto a well for the formation of a lipid bilayer with a smaller basesurface area and a working electrode with a larger base surface area.

DETAILED DESCRIPTION

The invention can be implemented in numerous ways, including as aprocess; an apparatus; a system; a composition of matter; a computerprogram product embodied on a computer readable storage medium; and/or aprocessor, such as a processor configured to execute instructions storedon and/or provided by a memory coupled to the processor. In thisspecification, these implementations, or any other form that theinvention may take, may be referred to as techniques. In general, theorder of the steps of disclosed processes may be altered within thescope of the invention. Unless stated otherwise, a component such as aprocessor or a memory described as being configured to perform a taskmay be implemented as a general component that is temporarily configuredto perform the task at a given time or a specific component that ismanufactured to perform the task. As used herein, the term ‘processor’refers to one or more devices, circuits, and/or processing coresconfigured to process data, such as computer program instructions.

A detailed description of one or more embodiments of the invention isprovided below along with accompanying figures that illustrate theprinciples of the invention. The invention is described in connectionwith such embodiments, but the invention is not limited to anyembodiment. The scope of the invention is limited only by the claims andthe invention encompasses numerous alternatives, modifications andequivalents. Numerous specific details are set forth in the followingdescription in order to provide a thorough understanding of theinvention. These details are provided for the purpose of example and theinvention may be practiced according to the claims without some or allof these specific details. For the purpose of clarity, technicalmaterial that is known in the technical fields related to the inventionhas not been described in detail so that the invention is notunnecessarily obscured.

Nanopore membrane devices having pore sizes on the order of onenanometer in internal diameter have shown promise in rapid nucleotidesequencing. When a voltage potential is applied across a nanoporeimmersed in a conducting fluid, a small ion current attributed to theconduction of ions across the nanopore can be observed. The size of thecurrent is sensitive to the pore size.

A nanopore based sequencing chip may be used for DNA sequencing. Ananopore based sequencing chip incorporates a large number of sensorcells configured as an array. For example, an array of one million cellsmay include 1000 rows by 1000 columns of cells.

FIG. 1 illustrates an embodiment of a cell 100 in a nanopore basedsequencing chip. A membrane 102 is formed over the surface of the cell.In some embodiments, membrane 102 is a lipid bilayer. The bulkelectrolyte 114 containing protein nanopore transmembrane molecularcomplexes (PNTMC) and the analyte of interest is placed directly ontothe surface of the cell. A single PNTMC 104 is inserted into membrane102 by electroporation. The individual membranes in the array areneither chemically nor electrically connected to each other. Thus, eachcell in the array is an independent sequencing machine, producing dataunique to the single polymer molecule associated with the PNTMC. PNTMC104 operates on the analytes and modulates the ionic current through theotherwise impermeable bilayer.

With continued reference to FIG. 1 , analog measurement circuitry 112 isconnected to an electrode 110 covered by a thin film of electrolyte 108.The thin film of electrolyte 108 is isolated from the bulk electrolyte114 by the ion-impermeable membrane 102. PNTMC 104 crosses membrane 102and provides the only path for ionic current to flow from the bulkliquid to working electrode 110. The cell also includes a counterelectrode (CE) 116. The cell also includes a reference electrode 117,which acts as an electrochemical potential sensor.

In some embodiments, a nanopore array enables parallel sequencing usingthe single molecule nanopore-based sequencing by synthesis (Nano-SBS)technique. FIG. 2 illustrates an embodiment of a cell 200 performingnucleotide sequencing with the Nano-SBS technique. In the Nano-SBStechnique, a template 202 to be sequenced and a primer are introduced tocell 200. To this template-primer complex, four differently taggednucleotides 208 are added to the bulk aqueous phase. As the correctlytagged nucleotide is complexed with the polymerase 204, the tail of thetag is positioned in the barrel of nanopore 206. The tag held in thebarrel of nanopore 206 generates a unique ionic blockade signal 210,thereby electronically identifying the added base due to the tags'distinct chemical structures.

FIG. 3 illustrates an embodiment of a cell about to perform nucleotidesequencing with pre-loaded tags. A nanopore 301 is formed in a membrane302. An enzyme 303 (e.g., a polymerase, such as a DNA polymerase) isassociated with the nanopore. In some cases, polymerase 303 iscovalently attached to nanopore 301. Polymerase 303 is associated with anucleic acid molecule 304 to be sequenced. In some embodiments, thenucleic acid molecule 304 is circular. In some cases, nucleic acidmolecule 304 is linear. In some embodiments, a nucleic acid primer 305is hybridized to a portion of nucleic acid molecule 304. Polymerase 303catalyzes the incorporation of nucleotides 306 onto primer 305 usingsingle stranded nucleic acid molecule 304 as a template. Nucleotides 306comprise tag species (“tags”) 307.

FIG. 4 illustrates an embodiment of a process 400 for nucleic acidsequencing with pre-loaded tags. At stage A, a tagged nucleotide (one offour different types: A, T, G, or C) is not associated with thepolymerase. At stage B, a tagged nucleotide is associated with thepolymerase. At stage C, the polymerase is in close proximity to thenanopore. The tag is pulled into the nanopore by an electrical fieldgenerated by a voltage applied across the membrane and/or the nanopore.

Some of the associated tagged nucleotides are not base paired with thenucleic acid molecule. These non-paired nucleotides typically arerejected by the polymerase within a time scale that is shorter than thetime scale for which correctly paired nucleotides remain associated withthe polymerase. Since the non-paired nucleotides are only transientlyassociated with the polymerase, process 400 as shown in FIG. 4 typicallydoes not proceed beyond stage B.

Before the polymerase is docked to the nanopore, the conductance of thenanopore is ˜0.300 pico Siemens (300 pS). At stage C, the conductance ofthe nanopore is about 60 pS, 80 pS, 100 pS, or 120 pS corresponding toone of the four types of tagged nucleotides. The polymerase undergoes anisomerization and a transphosphorylation reaction to incorporate thenucleotide into the growing nucleic acid molecule and release the tagmolecule. In particular, as the tag is held in the nanopore, a uniqueconductance signal (e.g., see signal 210 in FIG. 2 ) is generated due tothe tag's distinct chemical structures, thereby identifying the addedbase electronically. Repeating the cycle (i.e., stage A through E orstage A through F) allows for the sequencing of the nucleic acidmolecule. At stage D, the released tag passes through the nanopore.

In some cases, tagged nucleotides that are not incorporated into thegrowing nucleic acid molecule will also pass through the nanopore, asseen in stage F of FIG. 4 . The unincorporated nucleotide can bedetected by the nanopore in some instances, but the method provides ameans for distinguishing between an incorporated nucleotide and anunincorporated nucleotide based at least in part on the time for whichthe nucleotide is detected in the nanopore. Tags bound to unincorporatednucleotides pass through the nanopore quickly and are detected for ashort period of time (e.g., less than 10 ms), while tags bound toincorporated nucleotides are loaded into the nanopore and detected for along period of time (e.g., at least 10 ms).

FIG. 5 illustrates an embodiment of a circuitry 500 in a cell of ananopore based sequencing chip. As mentioned above, when the tag is heldin nanopore 502, a unique conductance signal (e.g., see signal 210 inFIG. 2 ) is generated due to the tag's distinct chemical structures,thereby identifying the added base electronically. The circuitry in FIG.5 maintains a constant voltage across nanopore 502 when the current flowis measured. In particular, the circuitry includes an operationalamplifier 504 and a pass device 506 that maintain a constant voltageequal to V_(a) or V_(b) across nanopore 502. The current flowing throughnanopore 502 is integrated at a capacitor n_(cap) 508 and measured by anAnalog-to-Digital (ADC) converter 510.

However, circuitry 500 has a number of drawbacks. One of the drawbacksis that circuitry 500 only measures unidirectional current flow. Anotherdrawback is that operational amplifier 504 in circuitry 500 mayintroduce a number of performance issues. For example, the offsetvoltage and the temperature drift of operational amplifier 504 may causethe actual voltage applied across nanopore 502 to vary across differentcells. The actual voltage applied across nanopore 502 may drift by tensof millivolts above or below the desired value, thereby causingsignificant measurement inaccuracies. In addition, the operationalamplifier noise may cause additional detection errors. Another drawbackis that the portions of the circuitry for maintaining a constant voltageacross the nanopore while current flow measurements are made arearea-intensive. For example, operational amplifier 504 occupiessignificantly more space in a cell than other components. As thenanopore based sequencing chip is scaled to include more and more cells,the area occupied by the operational amplifiers may increase to anunattainable size. Unfortunatly, shrinking the operational amplifier'ssize in a nanopore based sequencing chip with a large-sized array mayraise other performance issues. For example, it may exacerbate theoffset and noise problems in the cells even further.

FIG. 6 illustrates an embodiment of a circuitry 600 in a cell of ananopore based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state. One of the possible statesof the nanopore is an open-channel state when a tag-attachedpolyphosphate is absent from the barrel of the nanopore. Another fourpossible states of the nanopore correspond to the states when the fourdifferent types of tag-attached polyphosphate (A, T, G, or C) are heldin the barrel of the nanopore. Yet another possible state of thenanopore is when the membrane is ruptured. FIGS. 7A and 7B illustrateadditional embodiments of a circuitry (700 and 701) in a cell of ananopore based sequencing chip, wherein the voltage applied across thenanopore can be configured to vary over a time period during which thenanopore is in a particular detectable state. In the above circuits, theoperational amplifier is no longer required.

FIG. 6 shows a nanopore 602 that is inserted into a membrane 612, andnanopore 602 and membrane 612 are situated between a cell workingelectrode 614 and a counter electrode 616, such that a voltage isapplied across nanopore 602. Nanopore 602 is also in contact with a bulkliquid/electrolyte 618. Note that nanopore 602 and membrane 612 aredrawn upside down as compared to the nanopore and membrane in FIG. 1 .Hereinafter, a cell is meant to include at least a membrane, a nanopore,a working cell electrode, and the associated circuitry. In someembodiments, the counter electrode is shared between a plurality ofcells, and is therefore also referred to as a common electrode. Thecommon electrode can be configured to apply a common potential to thebulk liquid in contact with the nanopores in the measurements cells. Thecommon potential and the common electrode are common to all of themeasurement cells. There is a working cell electrode within eachmeasurement cell; in contrast to the common electrode, working cellelectrode 614 is configurable to apply a distinct potential that isindependent from the working cell electrodes in other measurement cells.

In FIGS. 7A and 7B, instead of showing a nanopore inserted in a membraneand the liquid surrounding the nanopore, an electrical model 702representing the electrical properties of the nanopore and the membraneand an electrical model 714 representing the electrical properties ofthe working electrode are shown. Note in FIGS. 7A and 7B that therespective circuitry does not require an extra capacitor (e.g., n_(cap)508 in FIG. 5 ) to be fabricated on-chip, thereby facilitating thereduction in size of the nanopore based sequencing chip.

Electrical model 702 includes a capacitor 706 that models a capacitanceassociated with the membrane (C_(membrane)) and a resistor 704 thatmodels a resistance associated with the nanopore in different states(e.g., the open-channel state or the states corresponding to havingdifferent types of tags or molecules inside the nanopore). Electricalmodel 714 includes a capacitor 716 that models a capacitance associatedwith the working electrode. The capacitance associated with the workingelectrode is also referred to as an electrochemical capacitance(C_(electrochemical)). The electrochemical capacitanceC_(electrochemical) associated with the working electrode includes adouble-layer capacitance and may further include a pseudocapacitance.

FIG. 7C illustrates a double layer that is formed at any interfacebetween a conductive electrode and an adjacent liquid electrolyte. If avoltage is applied, electronic charges (positive or negative) accumulatein the electrode at the interface between the conductive electrode andadjacent liquid electrolyte. The charge in the electrode is balanced byreorientation of dipoles and accumulation of ions of opposite charge inthe electrolyte near the interface. The accumulation of charges oneither side of the interface between electrode and electrolyte,separated by a small distance due to the finite size of charged speciesand solvent molecules in the electrolyte, acts like a dielectric in aconventional capacitor. The term “double layer” refers to the ensembleof electronic and ionic charge distribution in the vicinity of theinterface between the electrode and electrolyte. FIG. 7D illustrates apseudocapacitance effect that can be formed, simultaneously with theformation of a double-layer as in FIG. 7C, at an interface between aconductive electrode and an adjacent liquid electrolyte. Apseudocapacitor stores electrical energy faradaically by electron chargetransfer between the electrode and the electrolyte. This is accomplishedthrough electrosorption, reduction-oxidation reactions, or intercalationprocesses.

FIG. 8 illustrates an embodiment of a process 800 for analyzing amolecule inside a nanopore, wherein the nanopore is inserted in amembrane. Process 800 may be performed using the circuitries shown inFIG. 6, 7A, or 7B. FIG. 9 illustrates an embodiment of a plot of thevoltage applied across the nanopore versus time when process 800 isperformed and repeated three times. The voltage across the nanoporechanges over time. The rate of the voltage decay (i.e., the steepness ofthe slope of the voltage across the nanopore versus time plot) dependson the cell resistance (e.g., the resistance of resistor 704 in FIG.7A). More particularly, as the resistances associated with the nanoporein different states (e.g., the states corresponding to having differenttypes of molecules inside the nanopore) are different due to themolecules' distinct chemical structure, different corresponding rates ofvoltage decay may be observed and thus may be used to identify themolecule in the nanopore.

FIG. 10 illustrates the plots of the voltage applied across the nanoporeversus time when the nanopore is in different states. Curve 1002 showsthe rate of voltage decay during an open-channel state. In someembodiments, the resistance associated with the nanopore in anopen-channel state is in the range of 100 Mohm to 20 Gohm. Curves 1004,1006, 1008, and 1010 show the different rates of voltage decaycorresponding to the four capture states when the four different typesof tag-attached polyphosphate (A, T, G, or C) are held in the barrel ofthe nanopore. In some embodiments, the resistance associated with thenanopore in a capture state is within the range of 200 Mohm to 40 Gohm.Note that the slope of each of the plots is distinguishable from eachother.

Allowing the voltage applied across the nanopore to decay over a timeperiod during which the nanopore is in a particular detectable state hasmany advantages. One of the advantages is that the elimination of theoperational amplifier, the pass device, and the capacitor (e.g., n_(cap)508 in FIG. 5 ) that are otherwise fabricated on-chip in the cellcircuitry significantly reduces the footprint of a single cell in thenanopore based sequencing chip, thereby facilitating the scaling of thenanopore based sequencing chip to include more and more cells (e.g.,incorporating millions of cells in a nanopore based sequencing chip).The capacitance in parallel with the nanopore includes two portions: thecapacitance associated with the membrane and the capacitance associatedwith the integrated chip (IC). Due to the thin nature of the membrane,the capacitance associated with the membrane alone can suffice toachieve the required RC time constant without the need for additionalon-chip capacitance, thereby allowing significant reduction in cell sizeand chip size.

Another advantage is that the circuitry of a cell does not suffer fromoffset inaccuracies because V_(p)re is applied directly to the workingelectrode without any intervening circuitry. Another advantage is thatsince no switches are being opened or closed during the measurementintervals, the amount of charge injection is minimized.

Furthermore, the technique described above operates equally well usingpositive voltages or negative voltages. Bidirectional measurements havebeen shown to be helpful in characterizing a molecular complex. Forexample, they can be used to correct for baseline drift arising fromAC-non-faradaic operation.

The ratio of the capacitance associated with the membrane (seeC_(membrane) 706 of FIGS. 7A and 7B) and the capacitance associated withthe working electrode (see C_(electrochemical) 716 of FIGS. 7A and 7B)may be adjusted to achieve optimal overall system performance. Increasedsystem performance may be achieved by reducing C_(membrane) whilemaximizing C_(electrochemical). For example, C_(membrane) is adjusted toachieve the required RC time constant without the need for additionalon-chip capacitance, thereby allowing a significant reduction in cellsize and chip size. C electrochemical is maximized such that theimpedance associated with C_(electrochemical) is close to an AC(alternating current) short circuit compared with the impedanceassociated with C_(membrane). C_(electrochemical) is also maximized suchthat the information signal measured by the circuitries shown in FIG. 6,7A, or 7B becomes more stable and that the spurious signal convoluted ontop of the information signal is minimized.

FIG. 11 illustrates an embodiment of a cell 1100 in a nanopore basedsequencing chip. In this embodiment, the ratio of the C_(membrane) andC_(electrochemical) may be adjusted by increasing C_(electrochemical),as will be described in greater detail below.

Cell 1100 includes a conductive or metal layer 1101. Metal layer 1101connects cell 1100 to the remaining portions of the nanopore basedsequencing chip. In some embodiments, metal layer 1101 is the metal 6layer (M6). Cell 1100 further includes a dielectric layer 1104 aboveconductive layer 1101. Dielectric layer 1104 forms the walls surroundinga well 1105 in which a working electrode 1102 is located at the bottom.In some embodiments, working electrode 1102 is made of materials thatare resistant to corrosion and oxidation for non-faradaic conduction.The top surface of dielectric layer 1104 may be silanized. Silanizationforms a hydrophobic layer 1120 above the top surface of dielectric layer1104. Well 1105 formed by the dielectric layer walls 1104 furtherincludes a film of salt solution 1106 above working electrode 1102.

As shown in FIG. 11 , a membrane is formed on top of dielectric layer1104 and spans across well 1105. For example, the membrane includes alipid monolayer 1118 formed on top of hydrophobic layer 1120. As themembrane reaches the opening of well 1105, the lipid monolayertransitions to a lipid bilayer 1114 that spans across the opening of thewell. A bulk electrolyte 1108 containing protein nanopore transmembranemolecular complexes (PNTMC) and the analyte of interest is placeddirectly above the well. A single PNTMC/nanopore 1116 is inserted intolipid bilayer 1114 by electroporation. Nanopore 1116 crosses lipidbilayer 1114 and provides the only path for ionic flow from bulkelectrolyte 1108 to working electrode 1102.

Cell 1100 includes a counter electrode (CE) 1110. Cell 1100 alsoincludes a reference electrode 1112, which acts as an electrochemicalpotential sensor. In some embodiments, counter electrode 1110 is sharedbetween a plurality of cells, and is therefore also referred to as acommon electrode. The common electrode can be configured to apply acommon potential to the bulk liquid in contact with the nanopores in themeasurements cells. The common potential and the common electrode arecommon to all of the measurement cells.

As discussed above, the ratio of C_(membrane) and C_(electrochemical) incell 1100 may be adjusted by increasing C_(electrochemical). Theelectrochemical capacitance (C_(electrochemical)) associated withworking electrode 1102 may be increased by increasing the thickness ofworking electrode 1102. In some embodiments, the thickness of workingelectrode 1102 ranges from 10 nanometers to 1 micron.

C_(electrochemical) may also be increased by maximizing the specificsurface area of the electrode. The specific surface area of workingelectrode 1102 is the total surface area of the electrode per unit ofmass (e.g., m²/kg), per unit of volume (e.g., m²/m³ or m⁻¹), or per unitof base area (e.g., m²/m²) As the specific surface area increases, theelectrochemical capacitance (C_(electrochemical)) increases, and agreater amount of ions can be displaced with the same applied potentialbefore the capacitor becomes charged. For example, the specific surfacearea of the working electrode may be increased by making the electrode“spongy.”

Another way to increase C_(electrochemical) is by increasing the basesurface area of working electrode 1102. For example, if the workingelectrode has a cylindrical shape, then the base surface area of thecylinder may be increased. In another example, if the working electrodehas a rectangular prism shape, then the base surface area of therectangular prism may be increased. However, cell 1100 has a drawback.Working electrode 1102 and lipid bilayer 1114 have the same (or similar)base surface area or cross sectional area. When the base surface area ofworking electrode 1102 is increased, the base surface areas of theopening of well 1105 and lipid bilayer 1114 are both increased as well.As a result, both C_(membrane) and C_(electrochemical) are increasedsimultaneously. In other words, to optimize the overall systemperformance, C_(membrane) cannot be reduced while maximizingC_(electrochemical) by adjusting the base area of well 1105 alone.

In the present application, a non-faradaic electrochemical cell fornucleic acid sequencing that has a smaller aperture opening to a wellfor the formation of a lipid bilayer with a smaller base surface areaand a working electrode with a larger base surface area is disclosed.The base surface area of the opening to the well (which is the same asthe base surface area of the lipid bilayer) and the top base surfacearea of the working electrode that is exposed to the electrolyte can beadjusted independently of each other. Therefore, the two base surfaceareas may be adjusted independently to provide the desired ratio betweenC_(membrane) and C_(electrochemical) for optimized cell performance. Alipid bilayer is formed above the working electrode, and the lipidbilayer spans across a smaller aperture opening with a smaller basesurface area than the top base surface area of the working electrodeexposed to the electrolyte. With a smaller lipid bilayer base surfacearea and a larger working electrode top base surface area, C_(membrane)can be reduced while maximizing C_(electrochemical).

FIG. 12 illustrates an embodiment of a non-faradaic electrochemical cell1200 for nucleic acid sequencing that has a smaller aperture opening toa chalice well for the formation of a lipid bilayer with a smaller basesurface area and a working electrode with a larger base surface area.Cell 1200 is one of the cells in a nanopore based sequencing chip. Inconstrast to cell 1100, C_(membrane) and C_(electrochemical) in cell1200 may be adjusted independently by adjusting the base surface area ofthe membrane and the base surface area of the working electrodeseparately. Cell 1200 includes a conductive or metal layer 1201. Metallayer 1201 connects cell 1200 to the remaining portions of the nanoporebased sequencing chip. In some embodiments, metal layer 1201 is themetal 6 layer (M6). Cell 1200 further includes a working electrode 1202and a dielectric layer 1203 above metal layer 1201. In some embodiments,the base surface area of working electrode 1202 is circular or octagonalin shape and dielectric layer 1203 forms the walls surrounding workingelectrode 1202. Cell 1200 further includes a dielectric layer 1204 aboveworking electrode 1202 and dielectric layer 1203. Dielectric layer 1204forms the insulating wall surrounding a lower section (1205A) of a well1205. In some embodiments, dielectric layer 1203 and dielectric layer1204 together form a single piece of dielectric. Dielectric layer 1203is the portion that is disposed horizontally adjacent to workingelectrode 1202, and dielectric layer 1204 is the portion that isdisposed above the working electrode. In some embodiments, dielectriclayer 1203 and dielectric layer 1204 are separate pieces of dielectricand they may be grown separately. Dielectric material used to formdielectric layers 1203 and 1204 includes glass, oxide, siliconmononitride (SiN), Silicon dioxide (SiO₂), and the like.

Cell 1200 further includes a hydrophilic layer 1220 (e.g., titaniumnitrate, TiN) and a hydrophobic layer 1222 above dielectric layer 1204.Hydrophilic layer 1220 and hydrophobic layer 1222 together form theinsulating wall surrounding an upper section (1205B) of well 1205.Hydrophilic layer 1220 and hydrophobic layer 1222 together form anoverhang above the lower section (1205A) of well 1205. Alternatively,hydrophilic layer 1220 is optional. Hydrophobic layer 1222 forms theinsulating wall surrounding upper section 1205B of well 1205.Hydrophobic layer 1222 forms an overhang above the lower section (1205A)of well 1205. In some embodiments, hydrophobic layer 1222 is formed bysilanization. Alternatively, dielectric material that is hydrophobicsuch as hafnium oxide and polycrystalline silicon (poly-Si) may be usedto form hydrophobic layer 1222. In some embodiments, hydrophobic layer1222 has a thickness of about 1.5 nanometer (nm). Hydrophobic layer 1222has a thickness between 100 anstroms to 2 microns. The interface betweenhydrophobic layer 1222 and hydrophilic layer 1220 facilitates theformation of a lipid bilayer. The lipid bilayer is formed at theinterface between hydrophobic layer 1222 and hydrophilic layer 1220.

The upper section 1205B of well 1205 has an opening 1205C above theworking electrode. In some embodiments, opening 1205C above the workingelectrode is circular and the base surface area of the opening isπ*(d/2)², where d is the diameter of the opening. FIG. 13 illustrates atop view of a plurality of circular openings 1302 of a plurality ofwells in a nanopore based sequencing chip. In some embodiments, opening1205C above the working electrode is octogonal in shape. The basesurface areas of opening 1205C and the upper section 1205B of well 1205,respectively, are smaller than the bottom base surface area of the lowersection 1205A of well 1205. As the lipid bilayer spans across opening1205C, a reduction in the base surface area of opening 1205C results ina reduction in the base surface area of the lipid bilayer and also thecapacitance associated with the lipid bilayer. The lower section 1205Aof well 1205 provides a large reservoir/chalice with a bottom basesurface area larger than that in the upper section 1205B of well 1205.An increase in the bottom base surface area of the lower section 1205Aof well 1205 increases the top base surface area of the electrode thathas direct contact with the electrolyte/salt solution 1206, therebyincreasing the electrochemical capacitance associated with the workingelectrode.

Inside well 1205, salt solution/electrolyte 1206 is deposited aboveworking electrode 1202. Salt solution 1206 may include one of thefollowing: lithium chloride (LiCl), sodium chloride (NaCl), potassiumchloride (KCl), lithium glutamate, sodium glutamate, potassiumglutamate, lithium acetate, sodium acetate, potassium acetate, calciumchloride (CaCl₂), strontium chloride (SrCl₂), Manganese chloride(MnCl₂), and magnesium chloride (MgCl₂). In some embodiments, saltsolution 1206 has a thickness of about three microns (μm). The thicknessof salt solution 1206 may range from 0-5 microns.

A bulk electrolyte 1208 containing protein nanopore transmembranemolecular complexes (PNTMC) and the analyte of interest is placeddirectly above the well. A single PNTMC/nanopore is inserted into thelipid bilayer by electroporation. The nanopore crosses the lipid bilayerand provides the only path for ionic flow from bulk electrolyte 1208 toworking electrode 1202. Bulk electrolyte 1208 may further include one ofthe following: lithium chloride (LiCl), sodium chloride (NaCl),potassium chloride (KCl), lithium glutamate, sodium glutamate, potassiumglutamate, lithium acetate, sodium acetate, potassium acetate, calciumchloride (CaCl₂), strontium chloride (SrCl₂), Manganese chloride(MnCl₂), and magnesium chloride (MgCl₂).

Cell 1200 includes a counter electrode (CE) 1210. Cell 1200 alsoincludes a reference electrode 1212, which acts as an electrochemicalpotential sensor. In some embodiments, counter electrode 1210 is sharedbetween a plurality of cells, and is therefore also referred to as acommon electrode. The common electrode can be configured to apply acommon potential to the bulk liquid in contact with the nanopores in themeasurements cells. The common potential and the common electrode arecommon to all of the measurement cells.

In some embodiments, working electrode 1202 is a titanium nitride (TiN)working electrode with increased electrochemical capacitance. Theelectrochemical capacitance associated with working electrode 1202 maybe increased by maximizing the specific surface area of the electrode.The specific surface area of working electrode 1202 is the total surfacearea of the electrode per unit of mass (e.g., m²/kg), per unit of volume(e.g., m²/m³ or m⁻¹), or per unit of base area (e.g., m²/m²). As thesurface area increases, the electrochemical capacitance of the workingelectrode increases, and a greater amount of ions can be displaced withthe same applied potential before the capacitor becomes charged. Thesurface area of working electrode 1202 may be increased by making theTiN electrode “spongy” or porous. The TiN sponge soaks up electrolyteand creates a large effective surface area in contact with theelectrolyte.

FIGS. 14A-E illustrate an embodiment of a process 1400 for constructinga non-faradaic electrochemical cell (e.g., cell 1200) that has a smalleraperture opening to a well for the formation of a lipid bilayer with asmaller base surface area and a working electrode with a larger basesurface area.

FIG. 14A illustrates step A of process 1400. In some embodiments, anoptional anti-reflective layer (e.g., TiN) 1214 is disposed on top of aconductive layer 1201 (e.g., M6). A layer of dielectric 1203 (e.g.,SiO₂) is disposed on top of conductive layer 1201 or the optionalanti-reflective layer 1214. The conductive layer includes circuitriesthat deliver the signals from the cell to the rest of the chip. Forexample, the circuitries deliver signals from the cell to an integratingcapacitor. In some embodiments, the layer of dielectric 1203 aboveconductive layer 1201 has a thickness of about 400 nm. The layer ofdielectric 1203 is etched to create a hole. The hole provides a spacefor growing a spongy and porous electrode (e.g., a spongy TiNelectrode). A spongy and porous TiN layer is deposited to fill the hole.The spongy and porous TiN layer is grown and deposited in a manner tocreate rough, sparsely-spaced TiN columnar structures or columns of TiNcrystals that provide a high specific surface area that can come incontact with an electrolyte. The layer of spongy and porous TiN layercan be deposited using different deposition techniques, including atomiclayer deposition, chemical vapor deposition, physical vapor deposition(PVD) sputtering deposition, and the like. The TiN layer may also bedeposited by PVD sputtering deposition. For example, titanium can bereactively sputtered in an N₂ environment or directly sputtered from aTiN target. The conditions of each of the deposition methods may betuned in such a way to deposit sparsely-spaced TiN columnar structuresor columns of TiN crystals. For example, when the TiN layer is depositedby DC (direct current) reactive magnetron sputtering from a titanium(Ti) target, the deposition system can be tuned to use a lowtemperature, low substrate bias voltage (the DC voltage between thesilicon substrate and the Ti target), and high pressure (e.g., 25 mT)such that the TiN can be deposited more slowly and more gently to formcolumns of TiN crystals. In some embodiments, the depth of the depositedTiN layer is about 1.5 times the depth of the hole. The depth of thedeposited TiN layer is between 500 angstroms to 3 microns thick. Thediameter or width of the deposited TiN layer is between 20 nm to 100microns. The excess TiN layer is removed. For example, the excess TiNlayer may be removed using chemical mechanical polishing (CMP)techniques. The remaining TiN deposited in the hole forms a spongy andporous TiN working electrode 1202. After working electrode 1202 isformed, a layer of dielectric 1204 (e.g., SiO₂) is deposited on top ofthe dielectric 1203 and working electrode 1202. In some embodiments, thedepth of dielectric 1204 is between 100 nm to 2 microns.

FIG. 14B illustrates step B of process 1400. A hydrophilic layer 1220(e.g., titanium nitrate, TiN) is deposited above dielectric 1204. Insome embodiments, hydrophilic layer 1220 has a thickness between 100 nmto 2 microns. A hydrophobic layer 1222 is deposited above hydrophiliclayer 1220. In some embodiments, hydrophobic layer 1222 is formed bysilanization. Alternatively, dielectric material that is hydrophobicsuch as hafnium oxide and polycrystalline silicon (poly-Si) may be usedto form hydrophobic layer 1222. In some embodiments, hydrophobic layer1222 has a thickness of about 1.5 nanometer (nm). Hydrophobic layer 1222has a thickness between 100 angstroms to 2 microns. The hydrophobiclayer and the hydrophilic layer provide two differentiallyfunctionalizable surfaces with different surface chemistry behaviors,thereby facilitating the formation of a lipid bilayer at the interfacebetween the two layers.

FIG. 14C illustrates step C of process 1400. A photoresist mask 1224 isdeposited above hydrophobic layer 1222. The photoresist mask 1224includes a pattern for etching the small aperture opening to a well. Insome embodiments, the width of the pattern is selected to etch a smallaperture opening with a width or diameter between 20 nm to 1 micron.

FIG. 14D illustrates step D of process 1400. Hydrophobic layer 1222,hydrophilic layer 1220, and dielectric 1204 are etched anisotropicallyusing reactive ion etching (RIE). The etching is tuned (e.g., by tuningthe etching time) to etch vertically close to but not reaching electrode1202. The etching creates an initial well with a small aperture openingfor the formation of the lipid bilayer.

FIG. 14E illustrates step E of process 1400. Dielectric layer 1204 isisotropically etched to create a larger reservoir/chalice with a basesurface area larger than that of the opening, enlarging the exposedelectrode area while keeping the opening of the well small. In someembodiments, diluted hydrogen fluoride (dHF) or buffered oxide etch(BOE) is used as the wet etchant. The wet etchant selectively etchesdielectric layer 1204 but not hydrophobic layer 1222 and hydrophiliclayer 1220. Dielectric layer 1204 is made of a material that isdifferentially etchable from hydrophobic layer 1222 and hydrophiliclayer 1220. The etching is tuned to etch horizontally about 200 nm fromthe edge of electrode 1202. The photoresist mask 1224 is then removed.

FIGS. 15A-E illustrate an embodiment of a process 1500 for constructinga non-faradaic electrochemical cell that has a smaller aperture openingto a well for the formation of a lipid bilayer with a smaller basesurface area and a working electrode with a larger base surface area.

FIG. 15A illustrates step A of process 1500. In some embodiments, anoptional anti-reflective layer (e.g., TiN) 1514 is disposed on top of aconductive layer 1501 (e.g., M6). A layer of dielectric 1503 (e.g.,SiO₂) is disposed on top of conductive layer 1501 or the optionalanti-reflective layer 1514. The conductive layer includes circuitriesthat deliver the signals from the cell to the rest of the chip. Forexample, the circuitries deliver signals from the cell to an integratingcapacitor. In some embodiments, the layer of dielectric 1503 aboveconductive layer 1501 has a thickness of about 400 nm. The layer ofdielectric 1503 is etched to create a hole 1518. The hole 1518 providesa space for growing an electrode, e.g., a spongy and porous electrode(e.g., a spongy TiN electrode) and a sacrifical layer.

FIG. 15B illustrates step B of process 1500. A spongy and porous TiNlayer 1502A is deposited to fill a portion of hole 1518. TiN layer 1502Afurther covers the vertical walls 1519 of hole 1518. TiN layer 1502Aalso covers the top surface of dielectric layer 1503. In someembodiments, the spongy and porous TiN layer is grown and deposited in amanner to create rough, sparsely-spaced TiN columnar structures orcolumns of TiN crystals that provide a high specific surface area thatcan come in contact with an electrolyte. The layer of spongy and porousTiN layer can be deposited using different deposition techniques,including atomic layer deposition, chemical vapor deposition, physicalvapor deposition (PVD) sputtering deposition, and the like. The TiNlayer may also be deposited by PVD sputtering deposition. For example,titanium can be reactively sputtered in an N₂ environment or directlysputtered from a TiN target. The conditions of each of the depositionmethods may be tuned in such a way to deposit sparsely-spaced TiNcolumnar structures or columns of TiN crystals. For example, when theTiN layer is deposited by DC (direct current) reactive magnetronsputtering from a titanium (Ti) target, the deposition system can betuned to use a low temperature, low substrate bias voltage (the DCvoltage between the silicon substrate and the Ti target), and highpressure (e.g., 25 mT) such that the TiN can be deposited more slowlyand more gently to form columns of TiN crystals. A layer of sacrificallayer 1516 is deposited. In some embodiments, sacrifical layer 1516 is alayer of tungsten or nickel. Sacrifical layer 1516 fills hole 1518 andcovers the portion of TiN layer 1502A that covers the top surface ofdielectric layer 1503.

FIG. 15C illustrates step C of process 1500. The excess sacrifical layer1516 and the excess TiN layer 1502A are removed, for example by chemicalmechanical polishing (CMP) techniques, to create a coplanar surface. Theremaining bottom TiN portion 1502C and the remaining vertical TiNportion 1502B form a spongy and porous TiN working electrode 1502. Thediameter or width of working electrode 1502 is between 20 nm to 100microns.

FIG. 15D illustrates step D of process 1500. A layer of dielectric 1504(e.g, SiO₂) is deposited on top of the dielectric 1503, workingelectrode 1502, and sacrifical layer 1516. In some embodiments, thedepth of dielectric 1504 is about 1 micron.

FIG. 15E illustrates step E of process 1500. A small aperture opening ondielectric layer 1504 is etched (e.g., using RIE) anisotropically toexpose a portion of the sacrifical layer 1516. In some embodiments, thewidth of the small aperture opening is between 20 nm to 1 micron.

FIG. 15F illustrates step F of process 1500. Sacrificial layer 1516 isisotropically etched to create a larger reservoir/chalice with a basesurface area larger than that of the opening 1505C, enlarging theexposed electrode area while keeping the opening of the well small.Sacrificial layer 1516 is made of a material that is differentiallyetchable from dielectric layer 1504. In some embodiments, sacrificiallayer 1516 is made of tungsten, and a selective etchant such as hot H₂O₂is used as the wet etchant. The wet etchant selectively etchessacrificial layer 1516 without damaging dielectric layer 1504 andelectrode 1502. The advantage of using a sacrificial layer 1516 is thatthe etching process is more robust and the process requires less tuningbecause over etching does not damage dielectric layer 1504 or workingelectrode 1502.

As shown in FIG. 15F, dielectric layer 1504 forms the insulating wallsurrounding an upper section 1505B of a well 1505. Dielectric layer 1504forms an overhang above a lower section (1505A) of well 1505. Dielectriclayer 1503 forms the insulating wall surrounding a lower section (1505A)of a well 1505. Working electrode 1502 has a planar portion 1502Cdisposed at the bottom of the well. Planar portion 1502C has a top basesurface area that is larger than the base surface area of opening 1505C.Working electrode 1502 has a vertical wall portion 1502B extendingperdicular from the planar portion 1502C at the peripheral of the planarportion 1502C. Vertical wall 1502B forms an electrode wall surroundingthe lower section 1505A of well 1505. Vertical wall 1502B is adjacent tothe insulating wall formed by dielectric layer 1503 and providesadditional surface area that has contact with the electrolyte, therebyincreasing the capacitance associated with the working electrode.

Although the foregoing embodiments have been described in some detailfor purposes of clarity of understanding, the invention is not limitedto the details provided. There are many alternative ways of implementingthe invention. The disclosed embodiments are illustrative and notrestrictive.

What is claimed is:
 1. A method of constructing a nanopore cell,comprising: forming a conductive layer; disposing a working electrodeabove the conductive layer; disposing a layer of dielectric materialover the working electrode; etching a well in the layer of dielectricmaterial over the working electrode, the well having a first portion anda second portion, wherein the first portion of the well has a firstdiameter and an opening that is configured to be covered by a membrane,wherein the second portion of the well extends to and exposes a portionof the working electrode, wherein the second portion has a seconddiameter that is greater than the first diameter, and wherein both thefirst portion and the second portion are configured to contact a liquiddisposed in the well.
 2. The method of claim 1, wherein the firstportion is differentially etchable from the second portion.
 3. Themethod of claim 1, further comprising disposing a layer of photoresistover the layer of dielectric material.
 4. The method of claim 1, whereinthe dielectric material comprises silicon dioxide.
 5. The method ofclaim 1, wherein the working electrode comprises titanium nitride.